Discovered just a decade ago, RNA interference (RNAi) is likely one of the earliest innovations of eukaryotic life, pre-dating the divergence of fungi, plants, and animals. RNAi encompasses the cellular response to exogenous double-stranded RNA (dsRNA) - delivered experimentally or generated during a viral infection - and the response to parasitic genetic elements, such as transposons and highly repeated sequences. We seek to understand how the biochemical details of the RNAi mechanism reflect these functions of RNAi. Our goal is to understand the biochemical basis and biological logic of RNAi in humans, using Drosophila and mammalian cells as models. A better understanding of the biochemical basis for RNAi will, of course, lead to better experimental RNAi tools. Such tools provide the underpinnings of effective somatic genetics and have become an essential part of academic and pharmaceutical gene discovery. Understanding the molecular details of RNAi will also help reveal how genomic stability is maintained in the presence of selfish genetic elements, which compose about half the human genome. Loss of genetic stability in the soma can lead directly to cancer, and, in the germ line, to birth defects. The core of our experimental strategy is to use Drosophila melanogaster as a model system, because flies combine outstanding genetic, biochemical, and phenotypic tools, and we will extend our findings in flies to mammals, using cultured mouse and human cells. In both flies and mammals, RNAi is initiated by the conversion of long double-stranded RNA into small interfering RNAs (siRNAs) by members of the Dicer family of double-stranded RNA-specific endonucleases. We will use quantitative biochemical and molecular tools to decipher the mechanism by which distinct Dicer enzymes catalyze siRNA synthesis. We will work to understand why some Dicer enzymes require ATP for activity whereas others do not, why only some appear to be processive, and why specific Dicer enzymes are restricted to produce discrete classes of small RNAs. The siRNAs produced by Dicer function in RISC, the protein-siRNA complex that mediates the functions of RNAi. We will work to understand how cells load siRNAs into Argonaute proteins, the core constituents of RISC. An elaborate cellular pathway for RISC assembly is beginning to emerge from biochemical and genetic experiments. What is the biological purpose of this pathway? Does functional diversification of small RNAs and of Argonaute proteins reflect discrete biological roles for these molecules? We will also continue our efforts to define how RISC works, with particular emphasis on its role as an RNA-directed RNA endonuclease. Finally, we will turn our attention to understanding the origins, mechanisms, and functions of repeat-associated small RNAs (rasiRNAs), the poorly understood, small RNA class responsible for silencing transposons and repetitive sequences, thereby ensuring genomic stability.